Second Advanced Training Course in

Thrombosis & Haemostasis

Course Manual 1 Second Advanced Training Course Manual.indd 1

2/27/14 10:23 AM

Second Advanced Training Course in

Thrombosis & Haemostasis

Table of Contents 4 Message from the Chairman 5 Meet the ISTH 6 Advanced Training Course Program

6

Thursday March 13



6

Friday March 14



7

Saturday March 15



8

Sunday March 16

9 Speakers 12 Program Abstracts

12 S1 & S4 Blood Coagulation and its Regulation by Anticoagulant Pathways

16 S2 Challenges in the Diagnosis and Management of the Hemophilias

17 S3 Diagnosis and Management of von Willebrand Disease



18 S5 Hemostasis in Patients with Impaired Liver Function



19 S6 How to Approach a Patient with Bleeding



21 S7 Venous Thrombosis: Manifestations, Diagnosis and Therapy



23 S8 Antiphospholipid Syndrome



24 S9 Women Issues and Thrombosis



26 S10 Novel Antithrombotic Drugs



28 S11 How to Approach a Patient with Confirmed Venous Thrombosis



30 S12 Perioperative Management in Patients with Risk for Thrombosis



32 S13 Platelet Function



33 S14 Heparin-Induced Thrombocytopenia (HIT)



35 S15 Immune Thrombocytopenias

39 S16 Diagnosis and Treatment of Inherited Platelet Disorders

40 S17 Diagnosis and Treatment of Acquired Platelet Disorders



44 S18 Disseminated Intravascular Coagulation (DIC)

46 Sponsors 47 General Information 49 ISTH Membership

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Andreas Greinacher

Message from the Chairman & the ISTH

Join Today!

Meet the ISTH

The mission the ISTH is to advance the understanding, prevention, diagnosis and treatment of thrombotic and bleeding disorders. Andreas Greinacher Chairman, ISTH Education Committee

On behalf of the International Society on Thrombosis and Haemostasis (ISTH), it is a pleasure to welcome you to the Second Advanced Training Course of the ISTH in Cascais, Portugal. The course is designed to provide the latest training in biological and clinical aspects of hemostasis and thrombosis. Over the next three days you will take part in an intense examination on the subjects of blood coagulation and bleeding disorders, platelets and venous thrombosis. We are privileged to have some of the leading scientists and clinicians in our field take part as speakers. All speakers will deliver focused lectures followed by ample time for discussion and close interaction with the participants. There will be plenty of time in the afternoon and evenings for interactive sessions relating to the analysis of the topics discussed during the day. You will all have a chance to meet and talk with the experts. We invite you to take advantage of this unique opportunity to actively interact with your fellow course participants and the faculty. We wish you a successful meeting and hope you enjoy the course! Sincerely,

The ISTH first and foremost stands for leading edge science. As a global membership community of specialists in bleeding and clotting disorders, we are dedicated to transformative scientific discoveries to advance clinical practice and improve the lives of millions of people worldwide.

Programs & Activities • Journal of Thrombosis and Haemostasis (JTH) • ISTH Biennial World Congresses • ISTH SSC Annual Meetings • E-learning Opportunities Year Round • Networking and Career Development • Educational and Training Courses • Fellowships and Travel Grants • Scientific Standardization and Nomenclature • Professional Capacity Building in Developing Countries

Become a Member of the ISTH ISTH membership is an important investment in your own future at every stage in your career. To learn more about the rewarding benefits of membership, visit us online at www.isth.org.

Andreas Greinacher Chairman, ISTH Education Committee

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5 2/27/14 10:23 AM

10:30 – 11:00

Coffee break & networking

Venous Thrombosis All sessions will be held in the Longshot/Bogey room located on the first floor of the hotel.

11: 00 – 13:00

Advanced Training Course Program

S7 Venous Thrombosis: Manifestations, Diagnosis and Therapy Questions and discussions

Sam Schulman Canada

S8 Antiphospholipid Syndrome Questions and discussions

Flip de Groot Netherlands

Thursday, March 13

13:00 – 14:00

Lunch & networking

Blood Coagulation And Bleeding Disorders

14:00 – 16:00

S9 Women Issues and Thrombosis Questions and discussions

16:00 – 16:30

Coffee break & networking

16:30 – 18:30

Interactive session with speakers and/or case presentations

18:30 – 20-:00

Dinner & networking

20:00 – 21:00

Meet the Expert

13:30 – 14:00

14:00 – 16:00

Welcome and introduction to the course

Andreas Greinacher Germany

S1 Current Concepts of the Coagulation System Questions and discussions

Björn Dahlbäck Sweden

S2 Challenges in the Diagnosis and Management of Hemophilias Questions and discussions

David Lillicrap Canada

Saturday, March 15

16:00 – 16:30

Coffee break & networking

16:30 – 18:30

S3 Diagnosis and Management of von Willebrand Disease Questions and discussions

David Lillicrap Canada

S4 Control of Coagulation Questions and discussions

Björn Dahlbäck Sweden

18:30 – 20:00

Dinner & networking

20:00 – 21:00

Meet the Experts

Venous Thrombosis

Friday, March 14

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08:30 – 10:30

10:30 – 11:00

Blood Coagulation And Bleeding Disorders 08:30 – 10:30

Sabine Eichinger Austria

S10 Novel Antithrombotic Drugs Questions and discussions

Sam Schulman Canada

S11 How to Approach a Patient with Venous Thrombosis Questions and discussions

Bernd Pötzsch Germany

S12 Perioperative Management in Patients with Risk for Thrombosis Questions and discussions

Sabine Eichinger Austria

Coffee break & networking

Platelets, Platelet Disorders

S5 Hemostasis in Patients with Impaired Liver Function Questions and Discussions

Flip de Groot Netherlands

S6 How to Approach a Patient with Bleeding Questions and Discussions

Bernd Pötzsch Germany

11:00 – 13:00

S13 Platelet Function Questions and discussions

Christian Gachet France

S14 Heparin-induced Thrombocytopenia Questions and discussions

Ted Warkentin Canada

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Speakers

Second Advanced Training Course in

Thrombosis & Haemostasis

Björn Dahlbäck, Ph.D. 13:00 – 14:00

Lunch & networking

14:00 – 16:00

S15 Diagnosis and Management of Immune Thrombocytopenias Questions and discussions

Andreas Greinacher Germany

S16 Diagnosis and Treatment of Inherited Platelet Disorders Questions and discussions

Christian Gachet France

16:00 – 16:30

Coffee break & networking

16:30 – 18:30

Interactive session with speakers and/ or case presentations

18:30 – 20:00

Dinner & networking

20:00 – 21:00

Meet the Expert

Sabine Eichinger, M.D.

Sunday, March 16 Platelets, Platelet Disorders 08:00 – 10:00

S17 Diagnosis and Treatment of Acquired Platelet Disorders Questions and discussions

Björn Dahlbäck is professor of Blood Coagulation Research at Lund University, Department of Laboratory Medicine, Skåne University Hospital, Malmö, Sweden. His research on the molecular mechanisms of the protein C anticoagulant system has focused on the role of activated protein C and its cofactor protein S in the degradation of coagulation factors V and VIII. Observations made during these studies lead into studies of other biologically important host defense systems, in particular the complement system and the interactions between the coagulation and complement systems. He described that protein S in blood circulates both as free protein and bound to the complement regulatory protein C4b-binding protein (C4BP) and demonstrated that analysis of free protein S is the method of choice to detect inherited protein S deficiency. He has also been interested in the genetics of thrombophilia and described APC resistance, caused by FV Leiden as the major inherited risk factor of thrombosis. Recently, he was involved in the elucidation of a mysterious autosomal dominant bleeding disorder from East Texas. The disease is caused by a point mutation in factor V, which induces alternative splicing and the creation of a short form of FV that causes the bleeding tendency via tissue factor pathway inhibitor (TFPI). Björn Dahlbäck is member of the ISTH Council, the Swedish Royal Academy of Science, and honorary member of the American Society of Hematology. He has published 305 original papers and 99 reviews, has been cited approx. 22,000 times and has an H-index of 75. He has received many awards for his research.

Andreas Greinacher Germany

Sabine Eichinger is Associate Professor of Medicine at the Medical University of Vienna, Austria, and Head of the Anticoagulation Clinic at the Department of Medicine I of the Medical University Hospital in Vienna. Dr. Eichinger received her medical and scientific training at the Department of Medicine I, Division of Haematology and Haemostasis, University of Vienna, and at the Beth Israel Hospital, Harvard Medical School, Boston, USA. Dr. Eichinger’s anticoagulation research has included investigating the mechanism of action of pro-coagulants and anticoagulants in in vitro and in vivo models, and the pathophysiology of the coagulation system in women. She has undertaken research to evaluate the risk factors for thrombotic disease and biomarkers of the coagulation system. In 1992, she initiated one of the world’s largest studies in patients with venous thrombosis, the Austrian Study on Recurrent Venous Thromboembolism. She is the local principal investigator of several interventional studies in patients with venous thromboembolism and atrial fibrillation, and an internationally recognised expert in designing and conducting clinical studies. She is Chair-elect of the Scientific and Standardization Subcommittee (SSC) of the ISTH, and is the recipient of several academic awards.

Christian Gachet, M.D., Ph.D. S18 Disseminated Intravascular Coagulation Questions and discussions 10:00 – 10:30

Coffee break & networking

10:30 – 11:45

Interactive session with the speakers and/or case presentations

11:45 – 12:00

Course evaluation and farewell

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Ted Warkentin Canada

Dr. Christian Gachet graduated as a medical doctor in 1985 and gained his Ph.D. in Pharmacology in 1991, both at the Université Louis Pasteur, Strasbourg, France. He became a Research Director at INSERM (Institut National de la Santé et de la Recherche Médicale) in 1998. Dr. Gachet is currently the Director of the INSERM Research Unit 949 and the Scientific Director at Etablissement Français du Sang-Alsace. The focus of Dr. Gachet’s work has long been the molecular mechanisms of ADP-induced platelet activation and its inhibition by the thienopyridine compounds. This led to the characterization and the identification of some of the platelet P2 receptors. He carried out extensive work on the P2Y1 platelet receptor and the P2X1 receptor. Dr. Gachet’s current interest is in general platelet physiology (formation of blood platelets, megakaryopoiesis, platelet activation) and pharmacology (new drugs, new targets), animal models of platelet defects (BSS, MYH9, P2 receptors), mouse models of arterial thrombosis in a context of atherosclerosis and clinical studies on the variability of the response to clopidogrel. Dr. Gachet has published over 200 papers in peer-reviewed journals and over 300 communications in his area of expertise. He also serves on the editorial boards of the Journal of Thrombosis and Haemostasis(JTH). Dr. Gachet has received several distinctions for his work in the field of atherothrombosis.

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Speakers Andreas Greinacher, M.D.

Bernd Pötzsch, M.D.

Andreas Greinacher is an M.D. with a specialization in transfusion medicine and hemostasis. His scientific career is focused on platelet disorders, bridging immuno-hematology and hemostasis. He works at the University Hospital Greifswald, Germany, where he is head of the Institute of Immunology and Transfusion Medicine, the clinical thrombosis and hemostasis service, the hemostasis out-patient clinic, the transfusion and stem cell service and the immuno-hematology laboratory. Aside from heparininduced thrombocytopenia and drug dependent thrombocytopenia, he has a major interest in hereditary and acquired platelet disorders. His recent work is focusing on adopting nanotechnology and biophysical methods to investigate platelets and protein changes. He is section editor of several journals in the field of thrombosis and hemostasis and the current chairman of the education committee of the ISTH.

Bernd Pötzsch received his medical education at Justus-Liebig-University in Giessen, Germany, 1981 – 1988, and his postgraduate training at the Heart Centre Kerckhoff-Clinik in Bad Nauheim, Germany. Since 1999 he is a full-term professor and senior physician at the University of Bonn, Germany, specialized in hemostasis and thrombosis. His focus of research is pathogenesis, diagnosis and treatment of thrombophilia and use of aptamers in diagnosis and treatment of coagulation disorders.

Philip (Flip) G. de Groot, Ph.D.

Sam Schulman, M.D.

Phillip G. de Groot is a professor of Biochemistry appointed at the University Hospital in Utrecht, the Netherlands. Presently, he is deputy head of the department of Clinical Chemistry and Haematology. Besides his responsibility for laboratory diagnostics, particularly in the field of haematology, he has run a research group on thrombosis and haemostasis for over 30 years. He has published over 400 peer-reviewed articles and has a Hirsch index of 66. This year he was awarded with the biennial Distinguished Career Award of the ISTH. One of the major interests of his laboratory is to understand why the presence of antiphospholipid antibodies increases the risk on thrombosis. Their approach to this problem was twofold; they aim to develop assays available that identify the subpopulation of antiphospholipid antibodies responsible for the thrombotic risk and, secondly, to understand the physiological function of the major antigen in this syndrome, b2-glycoprotein I. If they understand the protein, they will understand why antibodies directed against this protein are one of the most common acquired risk factors for thrombosis and pregnancy morbidity.

Sam Schulman graduated from Karolinska Institute, Stockholm, Sweden in 1977 and became a specialist in Internal Medicine in 1984, with subspecialties in Haematology and in Coagulation in 1985. That year he also received his Dr Med Sc with the thesis: “Studies on the Medical Treatment of Deep Vein Thrombosis.” He has worked within the field of coagulation disorders continuously since 1984, worked as a consultant at the national Hemophilia Center at Tel Hashomer, Israel from 1992-1996, and was director of the Hemophilia Treatment Center in Stockholm from 1996-2004. His major research activities have been clinical studies in venous thromboembolism, including several randomized trials and in hemophilia and its complications. He is currently involved in trials with new antithrombotic agents, such as the oral thrombin inhibitors. He has been a member of the Executive Committee of the World Federation of Hemophilia (2000-2004) and was chairman of the Subcommittee on Control of Anticoagulation of the SSC Subcommittee of ISTH from 2005-2008. Dr. Schulman is associate professor in Internal Medicine at Karolinska Institute and since September 2004 also a professor in Medicine at McMaster University. He is Director of the Thrombosis Service at HHS-General Hospital in Hamilton and Director of the Clinical Thromboembolism Program of McMaster University.

David Lillicrap, M.D., FRCPC

Theodore (Ted) E. Warkentin M.D., BSc (Med), FRCP(C), FACP

David Lillicrap is a Professor in the Department of Pathology and Molecular Medicine at Queen’s University, Kingston, Canada. Since 2000, he has been the recipient of a Canada Research Chair in Molecular Hemostasis and is a past Career Investigator of the Heart and Stroke Foundation of Ontario. He has served on the Gene Therapy Working Group of the US National Hemophilia Foundation (NHF) and has been a member of NHF’s Medical and Scientific Advisory Committee. He is the Chair of the Research Committee of the World Federation of Hemophilia (WFH) and is a member of the Medical Advisory Board of the WFH. He recently completed a three-year term as the chairperson of the ISTH’s SSC Subcommittee on von Willebrand Factor, and is the current Chair of the Society’s SSC. He is an Associate Editor of the journal Blood, and a member of the editorial board of the British Journal of Haematology. His research program relates to molecular aspects of the hemostatic system with particular emphasis on novel therapeutic approaches and immunological complications of hemophilia A, and the genetics, biology and pathobiology of von Willebrand Factor.

Theodore E. Warkentin, M.D., is a Professor in the Department of Medicine and the Department of Pathology and Molecular Medicine at the Michael G. DeGroote School of Medicine, McMaster University, in Hamilton, Ontario, Canada. He is also Regional Director, Transfusion Medicine, of the Hamilton Regional Laboratory Medicine Program and Hematologist, Service of Clinical Hematology, at Hamilton Health Sciences, also in Hamilton. Dr. Warkentin received both his Bachelor of Science (in Medicine) and his M.D. from the University of Manitoba in Winnipeg, Manitoba, Canada. He completed a hematology research fellowship at McMaster University and postgraduate work in medicine and hematology at the University of Toronto and McMaster University, respectively. He was awarded the XVth Jean Julliard Prize at the XXIVth Congress of the International Society of Blood Transfusion (Makuhari, Japan; in 1996) and was a Research Scholar of the Heart and Stroke Foundation of Canada from 1993 to 1998. Dr Warkentin is the former Chair (four-year term ending 2007) of the Platelet Immunology SSC Subcommittee, and was the Chair of the 2004 and 2008 consensus conference guidelines on heparin-induced thrombocytopenia, written under the aegis of the American College of Chest Physicians (ACCP).

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Program Abstracts

Second Advanced Training Course in

Thrombosis & Haemostasis

(in program order)

Blood Coagulation and Bleeding Disorders S1 & S4 Blood Coagulation and its Regulation by Anticoagulant Pathways Björn Dahlbäck Lund University, Department of Laboratory Medicine Malmö, Sweden Primary haemostasis and blood coagulation have evolved as important defense mechanisms against bleeding. The initial occlusion of a vascular lesion by the platelet plug is temporally co-ordinated with the activation of coagulation. The coagulation pathway is carefully controlled by several anticoagulant mechanisms and under normal conditions they prevail over the procoagulant forces. Disturbances of the natural balance between the pro- and anticoagulant systems caused by genetic or acquired factors may result in bleeding or thrombotic diseases. Primary haemostasis mediated by platelet-protein interactions Damage of the vascular wall exposes blood to subendothelial tissue, which triggers the primary haemostasis events. Multiple coordinated interactions between receptors on platelets, plasma proteins, and tissue components result in the initial sealing of the wounded area. The platelet plug formation is the result of a series of reactions including adhesion, aggregation, release of granule content, and morphological changes. Adhesion is dependent on the interaction between platelets and the von Willebrand factor (VWF), a high molecular weight plasma protein composed of multiple disulphide-linked subunits. Freshly synthesized VWF multimers, which can be >20 million Da in mass and 4 um in length, undergo proteolytic processing in plasma mediated by the metalloprotease ADAMTS 13. In the adhesion process, VWF serves as a bridge between collagen in the subendothelium and platelet membrane glycoprotein Ib-V-IX (GP1b-V-IX). The adhesion process functions better under high shear stress, i.e. it is more efficient in small arteriole than in veins because high shear unfolds the VWF thus exposing the binding sites for GPIb-V-IX. Platelets also contain receptors for collagen (Integrin α2β1 and GPVI and GPVI) that enforce the anchoring of the platelets to the damaged tissue. Platelets undergo major morphological changes with rearrangement of the membrane and exposure of negatively charged phospholipids and formation of extensive pseudopodia that help anchor the platelets. Release of thromboxane A2, ADP, calcium, and serotonin results in additional platelet activation and contraction of smooth muscle cells of the vessel wall. A conformational change of the platelet Integrin αIIβ3 exposes binding sites for the adhesive proteins fibrinogen, VWF, fibronectin and thrombospondin, the bridging between platelets resulting in platelet aggregation. Activation and propagation of the blood coagulation system Generation of thrombin at sites of vascular injury is the result of a series of reactions referred to as blood coagulation. Thrombin is the key effector enzyme having important functions, including feedback amplification of coagulation by activating factor V (FV), factor VIII (FVIII), and factor XI (FXI). It also cleaves off fibrinopeptides A and B from fibrinogen, which results in the polymerization of fibrin monomers to a fibrin network, it activates the fibrin crosslinking factor XIII (FXIII), and it activates platelets by cleaving PAR-1 (protease activated receptor-1). Exposure of tissue factor (TF) to blood triggers the initiation of the coagulation system by binding plasma factor VII (FVII). TF is normally not in contact with blood but abundantly

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present in cells surrounding the vasculature. A small amount of FVII in plasma is activated (FVIIa) and the FVIIa-TF-complex converts factor IX (FIX) and factor X (FX) into active enzymes (FIXa and FXa). FIXa and FXa may remain bound to the TF-bearing cell or bind to the negatively charged phospholipid membrane of activated platelets. FXa and its cofactor activated FV (FVa) assemble on the activated platelets to form the prothrombinase complex that activates prothrombin to thrombin. FV can be activated directly by FXa but the majority of FV is activated by thrombin. Thrombin also activates FVIII to FVIIIa, which serves as a cofactor to FIXa in the tenase complex that activated FX to FXa. FVIII circulates bound to the VWF and is freed after activation to join FIXa on the platelet membrane in the formation of the tenase complex. The assembly of the prothrombinase and tenase complexes on the phospholipid surface is a prerequisite for the propagation of the coagulation system as highly efficient in converting several thousand substrate molecules per minute, whereas the free enzymes FXa and FIXa are inefficient (Fig. 1 schematically represents coagulation process).

Fig. 1 Activation of coagulation by TF on extravascular cells and propagation on platelets. The negative charge of the phospholipid membrane is due to the presence of phosphatidyl serine, which under normal conditions is located in the inner-layer leaflet of the cell membrane but it is translocated to the outer layer during platelet activation. All the participating proteins of the tenase and prothrombinase complexes have affinity for the negatively charged phospholipid surface, the enzymes and the substrates via their aminoterminal domains, which contain g-carboxy glutamic acid (Gla) residues. The formation of Gla is the result of a vitamin K-dependent post-translational modification of glutamic acid residues. The Gla-residues bind calcium, which is important for the correct folding of the Gla domain. Vitamin K-antagonists that are commonly used to treat thrombosis, inhibit the post-translational modification, resulting in misfolded Gla domains that are unable to bind negatively charged phospholipid membranes. Thrombin generation continues after the generation of the fibrin clot, which is important for activation of FXIII and the thrombin activatable fibrinolysis inhibitor (TAFI). Activated FXIII (FXIIIa) is a transglutaminase that catalyses covalent cross-linkage of fibrinogen. TAFI is a carboxypeptidase that removes the carboxy-terminal lysines from fibrin. As these lysines are important for the binding of fibrinolytic enzymes to the fibrin, TAFI inhibits fibrinolysis. Continued next page

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Blood Coagulation and Bleeding Disorders Recommended reading:

Continued: S1 & S4 Blood Coagulation and its Regulation by Anticoagulant Pathways

1.

Broze, GJ, Jr., and Girard, TJ. Tissue factor pathway inhibitor: structure-function. Front

The activation of coagulation via TF, which is referred to as the extrinsic pathway, is initiated in vivo in response to trauma. An alternative activation pathway involves the contact phase proteins factor XII, high-molecular weight kininogen (HMWK), prekallikrein and FXI and results in the generation of FXIa, that in turn activates FIX. These reactions are collectively called the intrinsic pathway and their physiological importance is not fully understood. The intrinsic pathway is not important in trauma-initiated coagulation because inherited deficiency of FXII is not associated with bleeding problems. FXI deficiency on the other hand yields a moderately severe bleeding disorder. However, several discoveries point to an important physiological function of the contact phase system. Thus, it was found that mice lacking FXII are protected against arterial thrombosis diseases including stroke and myocardial infarction. In addition, polyphosphates released from platelet granules have been identified as a possible physiological activator of the contact phase in vivo.

AT is intrinsically an inefficient serpin, but its activity is stimulated by heparin and by heparin-like molecules (heparan sulphates or chondroitine sulphates) that are present on the surface of endothelial cells. Homozygous antithrombin knockout mice have a lethal phenotype, demonstrating the importance of the protein for control of coagulation. The protein C anticoagulant system inhibits the cofactors FVIIIa and FVa (Fig. 2).

Biosci (Landmark Ed) 2012. 17:262-280. 2.

Broze, GJ, Jr., and Girard, TJ. Factor V, tissue factor pathway inhibitor, and east Texas bleeding disorder. J Clin Invest 2013. 123:3710-3712.

3.

Camire, RM, and Bos, MH. The molecular basis of factor V and VIII procofactor activation. J Thromb Haemost 2009. 7:1951-1961.

4.

Dahlback, B. Advances in understanding pathogenic mechanisms of thrombophilic disorders. Blood 2008. 112:19-27.

5.

Dahlback, B, and Villoutreix, BO. Regulation of blood coagulation by the protein C anticoagulant pathway: novel insights into structure-function

The plasma concentrations of the coagulation factors are very different concentrations, which relate to their specific functions in the pathway. In general, the early components have lower concentrations than those that take part at later stages. This is consistent with the principal organisation of the system with multiple reactions and amplification potential. Thus, the fibrinogen concentration (10 uM) is ≈50.000-fold higher than that of FVIII (0.2 nM). The high level of fibrinogen is required for the formation of the fibrin clot, whereas the low concentration of FVIII is more than sufficient to support FIXa in the activation of FX. Among the vitamin K-dependent proteins, FVII (10 nM) is the least abundant, FIX and FX being at intermediate levels (100 nM) and prothrombin circulating at the highest concentration (2 uM). Knockout mice technology have contributed to the elucidation of the relative importance of the various coagulation factors in vivo. The crucial importance of the TF pathway is demonstrated by the embryonic lethal phenotype associated with TF deficiency. In contrast, FVII-deficient mice develop normally in utero but succomb shortly after birth from severe bleeding. This difference in severity suggest a role for TF during embryogenesis beyond fibrin formation. Deficiencies of prothrombin and FV are associated with partial embryonic lethality and fatal haemorrhage. In contrast, FIX and FVIII deficient mice develop normally in utero but get haemophilia-like disease after birth. Mice deficient in fibrinogen suffer a moderate to severe bleeding phenotype similar to that of human fibrinogen deficiency. This suggests that thrombin generation is more important than fibrin deposition. Anticoagulant pathways regulating blood coagulation Coagulation is regulated at multiple levels by different anticoagulant principles such as enzyme inhibition and proteolytic degradation of cofactors FVa and FVIIIa. The initial steps of the TF pathway are controlled by tissue factor pathway inhibitor (TFPI). In humans, there are no deficiency states of TFPI described. Mice lacking TFPI have a lethal phenotype with uncontrolled activation of coagulation and consumption of coagulation factors. TFPI circulates a low concentration bound to FV. Recently, the bleeding phenotype of the autosomal dominant East Texas Bleeding disorder was found to be caused by increased plasma concentrations of TFPI. The increase in TFPI was caused by an alternatively spliced FV isoform (caused by a point mutation in the FV gene) that bound TFPI with high affinity causing retention of TFPI in the circulation. Several enzymes of the coagulation system are inhibited by the serine protease inhibitor (serpin) antithrombin (AT), which limits the coagulation process to sites of vascular injury.

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relationships and molecular recognition. Arterioscler Thromb Vasc Biol 2005. 25:1311-

Fig. 2 Activation of protein C and inhibition of the coagulation system by APC.

1320. 6.

Griffin, JH, Fernandez, JA, Gale, AJ, and Mosnier, LO. Activated protein C. J Thromb Haemost 2007. 5 Suppl 1:73-80.

The key component is protein C, a vitamin K-dependent zymogen, is activated by thrombin bound to thrombomodulin (TM) on the surface of intact endothelial cells. The endothelial protein C receptor (EPCR) stimulates the activation of protein C. The thrombin-mediated activation of protein C demonstrates that thrombin can function both as a pro- and an anticoagulant enzyme. The procoagulant functions are expressed at sites of vascular disrupture, whereas the anticoagulant functions require intact vessel walls with endothelial cells containing TM. Activated protein C (APC) cleaves peptide bonds in each of the membrane-bound cofactors FVa and FVIIIa. The anticoagulant activity of APC is stimulated by the cofactor protein S, a vitamin K-dependent plasma protein. Protein S exists in two forms in human plasma, approximately 30% as free protein, the remainder being bound to the complement regulatory protein C4b-binding protein (C4BP). Protein S is instrumental for the localization of the C4BP to negatively charged phospholipid membranes, which is a unique way to provide local complement regulatory activity. The free form of protein S functions as cofactor in degradation of both FVIIIa and FVa. In regulation of the tenase complex, not only protein S serves as cofactor but also the intact form of FV, which works in synergy with protein S as APC cofactor. Thus, FV like thrombin has both pro- and anticoagulant functions.

7.

Lenting, PJ, Casari, C, Christophe, OD, and Denis, CV. von Willebrand factor: the old, the new and the unknown. J Thromb Haemost 2012. 10:2428-2437.

8.

Morrissey, JH, Choi, SH, and Smith, SA. Polyphosphate: an ancient molecule that links platelets, coagulation, and inflammation. Blood 2012. 119:5972-5979.

9.

Morrissey, JH, Tajkhorshid, E, Sligar, SG, and Rienstra, CM. Tissue factor/factor VIIa complex: role of the membrane surface. Thromb Res 2012. 129 Suppl 2:S8-10.

10. Mosesson, MW. Fibrinogen and fibrin structure and functions. J Thromb Haemost 2005. 3:1894-1904. 11. Renne, T, Schmaier, AH, Nickel, KF, Blomback, M, and Maas, C. In vivo roles of factor XII. Blood 2012. 120:4296-4303.

The physiological importance of the protein C system is demonstrated by the severe thromboembolic disease that is associated with homozygous deficiency of protein C in both man and mice. Mice lacking the protein C or TM genes are affected by a lethal phenotype, the TM deficiency being particularly severe affecting the embryogenesis even before development of a functional cardiovascular system.

12. Sadler, JE. Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura. Blood 2008. 112:11-18. 13. Segers, K, Dahlback, B, and Nicolaes, GA. Coagulation factor V and thrombophilia: background and mechanisms. Thromb Haemost

APC also has anti-inflammatory and anti-apoptotic properties. These affects are dependent on the binding of APC to the endothelial protein C receptor (EPCR), which changes the substrate specificity of APC. When bound to EPCR, APC can cleave PAR-1 but is less efficient in cleaving FVa and FVIIIa. 

2007. 98:530-542. 14. Vincent, LM, Tran, S, Livaja, R, Bensend, TA, Milewicz, DM, and Dahlback, B. Coagulation factor V(A2440G) causes east Texas bleeding disorder via TFPIalpha. J Clin Invest 2013. 123:3777-3787.

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Blood Coagulation and Bleeding Disorders S2 Challenges in the Diagnosis and Management of the Hemophilias

S3 Diagnosis and Management of von Willebrand Disease David Lillicrap

David Lillicrap Queen’s University, Department of Pathology and Molecular Medicine Kingston, Ontario, Canada

Queen’s University, Department of Pathology and Molecular Medicine Kingston, Ontario, Canada

Hemophilia A and B are the two most common forms of severe inherited bleeding disease with incidence rates of approximately 1 in 5,000 and 1 in 25,000 male births, respectively. Both conditions are transmitted as X-linked recessive traits, with the majority of affected subjects being males. However, due to variations in the pattern of X inactivation, approximately 20% of female carriers of hemophilia can also express a mild bleeding tendency, often manifest as menorrhagia.

The index case of von Willebrand disease was described on one of the Aland Islands in the Baltic Sea in 1926. This teenage girl bled to death with her 4th menstrual period. Subsequent investigation of this family and others in the Aland archipelago, some 80 years later, demonstrated that they were affected by type 3 von Willebrand disease (VWD). von Willebrand disease is the most common inherited bleeding disorder in humans, with a prevalence of symptomatic subjects of approximately 1 in 1,000. In all documented VWD populations, females outnumber males by 2:1 due presumably to the enhanced likelihood of manifesting excessive mucocutaneous bleeding at the time of menses and childbirth. There are three subtypes of VWD: type 1 disease is a quantitative deficiency of functionally normal VWF. In most populations this accounts for ~65% of VWD cases. Type 2 VWD represents a group of qualitative VWF variants (types 2A, 2B, 2M and 2N) comprising approximately 30% of VWD and finally, type 3 VWD is the virtual absence of VWF (with accompanying very low levels of FVIII) occurring in approximately 1 in 1 million of the population.

The diagnosis of hemophilia is made through a combination of clinical and laboratory features. In approximately 60% of cases there will be a preceding family history of the condition. The clinical manifestations of hemophilia are very similar for deficiencies of factor VIII (FVIII) and factor IX (FIX), although there is some suggestion that severe FIX deficiency is less problematic than severe hemophilia A. Severe disease (factor levels 500 index cases of type 1 VWD indicate that only 65% of cases have candidate mutations in the coding regions and splice junctions of the VWF gene, and the pathogenic nature of some of these variants remains in question. Thus, the application of molecular analysis for the routine diagnosis of type 1 VWD is currently not recommended. The prevention and treatment of bleeding in VWD has changed very little over the past two decades. Many cases of type 1 VWD and some type 2 cases can be treated with desmopressin, and the remaining cases will require infusion with plasma-derived VWFFVIII concentrates. 

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Blood Coagulation and Bleeding Disorders S5 Hemostasis in Patients with Impaired Liver Function Philip G. de Groot Laboratory of Clinical Chemistry and Haematology, University Medical Center Utrecht, the Netherlands Chronic liver disease is a major cause of mortality and morbidity in many countries. Chronic or acute liver failure results in substantial changes in the hemostatic system. The liver is involved in the synthesis of most of the clotting factor proteins and reduced amounts of these proteins are found in the circulation with the exception of factor VIII. Moreover, the liver has lost partly its capacity to clear activated clotting factors-inhibitor complexes. Liver failure also results in a reduced platelet count and platelet function. All these defects are counterbalanced by a concomitant defect in anticoagulant and pro-fibrinolytic factors. Moreover, a decreased platelet function is counterbalanced by elevated levels of von Willebrand factor. The classic assays to detect a bleeding disorder, the prothrombin time (PT) and the activated partial thromboplastin time (APTT) do not correlate with a bleeding tendency because these assays do not measure the reduced activity of the physiological inhibitors such as antithrombin. The thrombin generation assay, an assay that is sensitive for these inhibitors, is often within a normal range in patients with liver cirrhosis, indicating that in these patients the hemostasic system is rebalanced. This rebalance is represented by a limited bleeding during surgery, including liver transplantation and by the thrombotic complications regularly seen after surgery. In this lecture I will discuss this rebalance and explain that this balance is less stable. The balance easily tip towards a hyper-or hypocoagulable state. Coagulopathy in patients with critical liver dysfunction is complex and can quickly decompensate to bleeding as well as to thrombosis. Liver cirrhosis is a unique clinical setting in which bleeding and thrombosis coexist. 

Recommended reading: 1.

Lisman T, Leebeek FW & de Groot PG. (2002) Haemostatic abnormalities in patients with liver disease. J Hepatol 37: 280-287.

2.

Lisman T, Caldwell SH, Burroughs AK, Northup PG, Senzolo M, Stravitz RT, Tripodi A, Trotter JF, Valla DC, Porte RJ; Coagulation in Liver Disease Study Group (2010) Hemostasis and thrombosis in patients with liver disease: the ups and downs. J Hepatol. 53: 362-71.

3.

Nowatari T, Murata S, Fukunaga K, Ohkohchi N. (2013) Role of platelets in chronic liver disease and acute liver injury. Hepatol Res. Jul 11 [Epub ahead of print].

4.

Arshad F, Lisman T, Porte RJ.(2013) Hypercoagulability as a contributor to thrombotic complications in the liver transplant recipient. Liver Int. 33: 820-7.

5.

Northup PG, Argo CK, Shah N, Caldwell SH (2012) Hypercoagulation and thrombophilia in nonalcoholic fatty liver disease: mechanisms, human evidence, therapeutic implications, and preventive implications. Semin Liver Dis. 32: 39-48.

6.

Tripodi A, Primignani M, Chantarangkul V, et al. (2009) An imbalance of pro- vs anticoagulation factors in plasma from patients with cirrhosis. Gastroenterology 137: 2105– 11.

S6 How to Approach a Patient with Bleeding Bernd Pötzsch Institute of Experimental Hematology and Transfusion Medicine University Hospital Bonn Bonn, Germany Bleeding disorders can be inherited or acquired and include coagulation factor deficiencies, hyperfibrinolysis, platelet deficiencies and/or dysfunctions, and von Willebrand´s disease (vWD). The initial evaluation of a patient with a suspected bleeding disorder should include a comprehensive medical and bleeding history, a complete family history, a detailed physical examination and selected laboratory tests. The bleeding history may provide important clues about the likelihood of a bleeding disorder and the type of the bleeding disorder. For example mucocutaneous bleeding such as petechiae, bruising, epistaxis, gastrointestinal bleeding and/or menorrhagia suggests disorders of platelets, von Willebrand factor (vWF) or a vascular bleeding disorder whereas bleeding into muscles and joints, soft tissues and delayed surgical bleeding suggests disorders of coagulation factors. The use of standardized scores to quantitate bleeding disorders is recommended. Standardized and validated bleeding questionnaires are available (Biss 2010). Physical examination should evaluate the localization, size, and age of hematomas and the presence of any signs of bleeding such as hemarthroses or evidence of chronic joint abnormalities. Signs of coexisting illness may be indicative for acquired bleeding disorders. For example lymphadenopathy and/or organomegaly suggest an infiltrative process such as malignancy while signs of liver failure suggest acquired coagulation factor deficiencies. Initial tests to screen for bleeding disorders should include a complete blood count (CBC), blood film, whole blood platelet function testing, prothrombin time (PT), activated partial thromboplastin time (APTT), and factor XIII testing. The CBC is performed to exclude thrombocytopenia and to detect additional pathologies of white blood cells and red cells. The blood film provides further information regarding platelet and leukocyte morphology. Several point-of-care tests measuring the platelet function in whole blood are commercially available. For example the platelet function analyzer (PFA) provides a simple and rapid assessment of high shear-dependent platelet function. This test is successfully used in the screening for vWD but lacks sensitivity for some congenital platelet disorders such as patients with P2Y12 deficiency and patients with granule deficiencies. The PT and APTT measure the activity of all coagulation factors that are involved in the generation of thrombin and thrombin-dependent fibrin formation. Clotting times of both assays within the age-specific reference ranges make the presence of a clinical relevant clotting factor deficiency unlikely although it should be noted that an aPTT within the reference range does not reliably exclude mild FVIII, FIX or FXI deficiency. Therefore, factor assays should be performed if the bleeding history or the family history suggests a mild bleeding disorder. Testing of FXIII activity is included into the laboratory screen because the PT and aPTT are not sensitive for FXIII. Continued next page

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Blood Coagulation and Bleeding Disorders

Continued: S6 How to Approach a Patient with Bleeding

Based on the initial test findings, plus the degree of clinical evidence, further evaluation may or may not be required. A negative bleeding history together with screening tests within the reference ranges make the presence of a bleeding disorder most unlikely and no further testing is recommended. Abnormal results of the initial screen require additional testing. For example, a prolonged closure time in the PFA together with a slightly prolonged APTT requires further testing for vWD including vWF-Ag ELISA and functional vWF assays. In patients with a positive bleeding history and no evidence of pathological laboratory tests it is difficult to establish a final diagnosis. In those cases a diagnosis of a bleeding disorder of unknown causes should be made. 

Recommended reading: 1.

Rodeghiero E, Kadir RA, Tosetto A, James PD. Relevance of quantitative assessment of bleeding in haemorrhagic disorders. Haemophilia 2008; 14 (suppl 3): 68

2.

Biss TT, Blanchette VS, Clark DS, Bowman M, Wakefield CD, Silva M, Lillicrap D, James PD, Rand ML. Quantitation of bleeding symptoms in children with von Willebrand disease: use of a standardized pediatric bleeding questionnaire. J Thromb Haemost 2010; 8: 950

3.

Harrison P, Mackie I, Mumford A, Briggs C, Liesner R, Winter M, Machin S. Guidelines for the laboratory investigation of heritable disorders of platelet function. Br J Haematol 2011; 155: 30

4.

Hayward CP. Diagnosis and management of mild bleeding disorders. Hematology Am Soc Hematol Educ Program 2005; 2005: 423

Venous Thrombosis S7 Venous Thrombosis: Manifestations, Diagnosis and Therapy Sam Schulman McMaster University, Clinical Thromboembolism Program Hamilton, Ontario, Canada The incidence of venous thromboembolism (VTE) has been reported from many population studies as approximately 100 per 100,000 but is influenced by selection of adults or certain age range and the use of autopsy. The incidence has often been quoted as lower in Asia, but there may be confounding by suspicion bias. The best estimate of difference between races is derived from a very large population cohort in California, demonstrating a decreasing rate from African Americans via whites and Hispanics to the lowest among Asian and Pacific islanders. Whereas there is a well-described exponential increase with age, the influence by sex is controversial. Most cohort data indicate a higher risk for young women and possibly for old men. A seasonal variation with peaks in December-January has been ascribed to variations in the fibrinogen level; in turn a possible result of respiratory tract infections. More specifically, the incidence of deep vein thrombosis (DVT) is often stated to be 50100% higher than that of pulmonary embolism (PE). This is logical since PE is considered as almost always originating from the leg veins. Patients with venographically confirmed DVT have asymptomatic pulmonary embolism on lung-scan in 30-50% whereas 70% of carefully investigated patients with PE turn out to have silent DVT. Typical, yet unspecific symptoms of DVT are pain deep in the calf or thigh, and unilateral swelling. Increased skin temperature, tenderness and discoloration are even less specific. In case of essentially total obstruction of venous return the leg becomes cyanotic and very painful – phlegmasia cerulea dolens – and with massive edema a compartment syndrome with closure also of the arterial circulation the leg becomes pale and very painful – phlegmasia alba dolens. Both conditions may lead to loss of the limb. For PE the most common symptoms in decreasing order are pleuritic pain (65%), dyspnea (20%), syncope (10%) and hemoptysis (few cases). The diagnosis of VTE using clinical symptoms and signs has low sensitivity and specificity. A clinical probability assessment is, however, valuable as part of a diagnostic algorithm. For low clinical probability a negative D-dimer test usually excludes the diagnosis. For high clinical probability, imaging diagnostic techniques are required, typically compression ultrasound for suspected DVT and computed tomography of pulmonary arteries for suspected PE, although in some cases ventilation-perfusion scan is advantageous. The standard initial treatment today is subcutaneous low-molecular-weight heparin (LMWH) without monitoring. Patients with DVT can generally be managed as outpatients. For those with PE a risk stratification tool should be used to select for outpatient treatment. A vitamin K antagonist should be started simultaneously, overlapping at least 5 days and then continue for 3-6 months. At that point an assessment of risk vs. benefit of long-term anticoagulation should be performed. For patients with massive PE and hemodynamic instability thrombolytic therapy is indicated. Patients with massive DVT should be assessed for catheter-directed thrombolysis +/- mechanical removal. Over 10 years about 30% of patients with unprovoked VTE will have a recurrence and DVT will almost always recur as lower extremity thrombosis. Conversely, PE usually recurs as PE. (Continued next page)

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Venous Thrombosis S8 Antiphospholipid Syndrome S7 Venous Thrombosis: Manifestations, Diagnosis and Therapy (Continued)

Philip G. de Groot

Case fatality is problematic to assess and influenced by the study methodology and autopsy rates. It is higher in PE, at least in the short-term perspective. At least 50, possibly 80% of patients with DVT develop venous insufficiency as part of the post-thrombotic syndrome and the severe form with venous ulcers has a linear increase, reaching 5% at 10 years. The mortality in patients with VTE is higher than in controls matched for age and sex. It is also specifically higher than expected for cancer or myocardial infarction and ischemic stroke. Finally, the prevalence of VTE is projected to double until 2050.  Recommended reading: 1.

Heit JA, Silverstein MD, Mohr DN, Petterson TM, Lohse CM, O’Fallon WM, Melton LJ, 3rd. The epidemiology of venous thromboembolism in the community. Thromb Haemost. 2001; 86: 452-63.

1.

Giannakopoulos B, Krilis SA. (2013) The pathogenesis of the antiphospholipid syndrome N. Engl. J. Med. 368: 1033-44

2.

de Groot PG, Urbanus RT (2012) The significance of auto-antibodies against β2Glycoprotein I. Blood 120: 266-74

3.

de Groot PG, Meijers JC. (2011) β(2) -Glycoprotein I: evolution, structure and function. J. Thromb. Haemostas. 9: 12751284.

4.

Ruiz-Irastorza G, Cuadrado MJ, Ruiz-Arruza I, Brey R, Crowther M, Derksen R, Erkan D, Krilis S, Machin S, Pengo V, Pierangeli S, Tektonidou M, Khamashta M. (2011) Evidence-based recommendations for the prevention and long-term management of thrombosis in antiphospholipid antibodypositive patients: report of a task force at the 13th International Congress on antiphospholipid antibodies. Lupus 20: 206-18

Prandoni P, Lensing AW, Cogo A, Cuppini S, Villalta S, Carta M, Cattelan AM, Polistena P, Bernardi E, Prins MH. The long-term clinical course of acute deep venous thrombosis. Ann Intern Med. 1996; 125: 1-7.

3.

Schulman S, Lindmarker P, Holmstrom M, Lärfars G, Carlsson A, Nicol P, Svensson E, Ljungberg B, Viering S, Nordlander S, Leijd B, Jahed K, Hjorth M, Linder O, Beckman M. Post-thrombotic syndrome, recurrence, and death 10 years after the first episode of venous thromboembolism treated with warfarin for 6 weeks or 6 months. J Thromb Haemost. 2006; 4: 734-42.

4.

Spencer FA, Emery C, Lessard D, Anderson F, Emani S, Aragam J, Becker RC, Goldberg RJ. The Worcester Venous Thromboembolism study: a population-based study of the clinical epidemiology of venous thromboembolism. J Gen Intern Med. 2006; 21: 722-7.

5.

Kearon C, Akl EA, Comerota AJ, et al. Antithrombotic therapy for VTE disease: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012;141:e419S-94S.

5.

6.

Holbrook A, Schulman S, Witt DM, et al. Evidence-based management of anticoagulant therapy. Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012;141:e89S-119S.

de Groot PG, Urbanus RT (2012) The future of antiphospholipid antibody testing. Sem. Thromb. Hemostas. 38: 412-20.

6.

7.

Garcia DA, Baglin TP, Weitz JI, et al. Parenteral anticoagulants. Antithrombotic therapy and prevention of thrombosis, 9th ed: American College of Chest Physicians evidence-based clinical practice guidelines. Chest. 2012;141:e24S-e43S.

Barbhaiya M, Erkan D. (2013) Top 10 clinical research developments in antiphospholipid syndrome. Curr Rheumatol Rep. 15: 367

8.

Wells, PS, Anderson, DR, Rodger, M, et al. (2003). Evaluation of D-dimer in the diagnosis of suspected deep-vein thrombosis. N Engl J Med 349, s. 1227-35.

9.

Wells, PS, Anderson, DR, Rodger, M, et al. (2000). Derivation of a simple clinical model to categorize patients probability of pulmonary embolism: increasing the models utility with the SimpliRED D-dimer. Thromb Haemost 83, s. 416-20.

11. Prandoni P, Bilora F, Marchiori A, Bernardi E, Petrobelli F, Lensing AW, et al. An association between atherosclerosis and venous thrombosis. N Engl J Med 2003;348:1435-1441.

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Recommended reading:

2.

10. Bates SM, Jaeschke R, Stevens SM, Goodacre S, Wells PS, Stevenson MD, et al. Diagnosis of DVT: Antithrombotic therapy and prevention of thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012;141:e351S-418S.

22

Laboratory of Clinical Chemistry and Haematology, University Medical Center Utrecht, the Netherlands

7.

Lockshin MD. (2013) Pregnancy and antiphospholipid syndrome. Am J Reprod Immunol. 69: 585-7

8.

Andreoli L, Chighizola CB, Banzato A, Pons-Estel GJ, de Jesus GR, Erkan D; on behalf of APS ACTION. (2013) The estimated frequency of antiphospholipid antibodies in patients with pregnancy morbidity, stroke, myocardial infarction, and deep vein thrombosis. Arthritis Care Res (Hoboken). Jul 16. [Epub ahead of print]

The antiphospholipid syndrome is an auto-immune disease characterized by thrombotic complications in both arteries and veins as well as fetal losses in combination with the presence of so-called antiphospholipid antibodies in plasma of these patients. Antiphospholipid antibodies are a family of auto-antibodies that can be measured with different assays that determine the presence of closely related but not overlapping antibody populations. Although the presence of these autoantibodies is regarded as a common cause of thrombosis and pregnancy morbidity in individuals at an age under 50 years, the true frequency of clinical significant antiphospholipid antibodies in not known. The lack of large-scale prospective population studies, the multi-factorial nature of thrombosis and fetal loss and the lack of standardization of the assays to detect the presence of these antibodies are major hurdles to determine the magnitude of the anti-phospholipid antibody problem. It is now generally accepted that the relevant auto-antibodies are not directed against negatively charged phospholipids but towards plasma proteins bound to these phospholipids. The most prominent antigen in APS is β2-Glycoprotein I (β2-GPI), a plasma protein with affinity towards anionic phospholipids. APS is an intriguing syndrome because we have difficulties to comprehend how the presence of auto-antibodies against β2-glycoprotein I increases the risk for thrombosis and fetal loss. β2-Glycoprotein I is a plasma protein without a clear function and individuals without this protein seem to be completely healthy. Moreover, the most relevant assay that we use to detect the presence of auto-antibodies against β2-glycoprotein I, a prolongation of a clotting assay named Lupus anticoagulant, express an opposite effect on coagulation as expected for a thrombotic risk. Prolongation of clotting assays points to a bleeding tendency, not a thrombotic tendency. Both the target of the auto-antibodies, β2-Glycoprotein I, and the detection method, lupus anticoagulant, do not give us a lead to the mechanism behind the increased thrombotic risk. Nevertheless, mouse models in which auto-antibodies against β2-glycoprotein I isolated from patients were used show abundantly clear that these auto-antibodies are the cause of the increased risk for thrombotic manifestations and pregnancy morbidity. In the present lecture I will introduce the antiphospholipid syndrome and explain the difficulties in the diagnosis of the syndrome. I will discuss the relative importance of the different assay we have available to diagnose the syndrome. I will give some possible explanations why individuals with these auto-antibodies in their blood have such a high risk of severe thrombotic complications at a younger age. I will finish with the different treatment options. 

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Venous Thrombosis S9 Women Issues and Thrombosis Sabine Eichinger Medical University of Vienna, Department of Internal Med Vienna, Austria Women are subject to specific hormonal changes which influence the coagulation and fibrinolytic systems and put them at increased thrombotic risk. During reproductive age use of hormone contraceptives, profertility ovarian stimulation or pregnancy alter the pro- and anticoagulant forces, while after menopause age-related aspects or hormone replacement therapy contribute to a hypercoagulable state. Hormone contraceptive use increases the risk of venous thromboembolism (VTE) about 2to 6-fold (1). The increased risk is related to the dose of estrogen but is also influenced by the type of progestogen (2). Non-oral hormonal contraceptives including the transdermal patch or the contraceptive vaginal ring are also associated with an increased risk of VTE (3). Evidence regarding the cardiovascular safety of progestogen-only methods of contraception is limited. A systematic review and meta-analysis of published data concluded that the use of progestogen-only contraceptives was not associated with an increased risk of VTE compared with non-users of hormonal contraception. However, the potential association between injectable progestogens and thrombosis requires further study (4).The relative risk of VTE compared with non-users among women using the levonorgestel-releasing intrauterine system (Mirena®) was low (RR 0.57 (95% CI; 0.41-0.81) (3). Ovarian hyper-stimulation does increase the risk of thromboembolic disorders and peaks dramatically in pregnant women with the ovarian hyper-stimulation syndrome requiring hospital admission. Pregnancy is a major risk factor for thrombosis. The risk of thrombosis is increased throughout pregnancy and is particularly high after delivery. Anticoagulant thromboprophylaxis is prescribed with analogy to prophylaxis outside pregnancy and is not standardized. Low-molecular-weight heparin (LMWH) is the drug of choice for preventing pregnancy-related VTE, whereas in puerperium the oral anticoagulants can be alternatively used. Women who are candidates for antithrombotic prophylaxis are those with a previous VTE or with a known severe inherited or acquired thrombophilia. Which type of thrombophilia does increase significantly the risk of VTE during pregnancy, suggesting the appropriateness of LMWH prophylaxis, is matter of debate. For the treatment of acute VTE in pregnant women fixed-dose, weight-adjusted subcutaneous LMWH is the anticoagulant of choice and should be given at a therapeutic dose throughout pregnancy (5). LMWH should be discontinued 24 hours before induction of labour or caesarean section, restarted at a reduced dose when it is safe to do so and continued for an additional six to eight weeks.

replacement therapy and a risk being greatest in users of oral formulations containing medroxyprogesterone acetate (7). Hormone replacement therapy also confers an increased risk of recurrent venous thrombosis. Oral hormone replacement therapy increases not only the risk of venous thrombosis but also of stroke (8). The risk is higher with advancing age and if additional risk factors, such as obesity, previous thromboembolic disease, smoking, and immobility are present. In otherwise healthy women younger than 60 years the absolute risk of thromboembolic disease is low. 

Recommended reading: 1.

van Hylckama Vlieg A, Middeldorp S. Hormone therapies and venous thromboembolism: where are we now? J Thromb Haemost 2011;9:257-66.

2.

Lidegaard Ø, Nielsen LH, Skovlund CW, Skjeldestad FE, Løkkegaard E. Risk of venous thromboembolism from use of oral contraceptives containing different progestogens and oestrogen doses: Danish cohort study, 2001-9. BMJ 2011;343:d6423.

3.

Lidegaard O, Nielsen LH, Skovlund CW, Løkkegaard E. Venous thrombosis in users of non-oral hormonal contraception: follow-up study, Denmark 2001-10. BMJ 2012;344:e2990.

4.

Mantha S, Karp R, Raghavan V, Terrin N, Bauer KA, Zwicker JI. Assessing the risk of venous thromboembolic events in women taking progestin-only contraception: a meta-analysis. BMJ. 2012;345:e4944

5.

Arya R. How I manage venous thromboembolism in pregnancy. Br J Haematol. 2011;153:698708

6.

Hickey M, Elliott J, Davison SL. Hormone replacement therapy. BMJ. 2012;344:e763

7.

Sweetland S, Beral V, Balkwill A, Liu B, Benson VS, Canonico M, Green J, Reeves GK; Million Women Study Collaborators1. Venous thromboembolism risk in relation to use of different types of postmenopausal hormone therapy in a large prospective study. J Thromb Haemost. 2012;10:2277-86

Menopause is accompanied by processes of physiological aging which is associated with increased plasma levels of many proteins of blood coagulation, alterations of platelets and fibrinolysis impairment. Hormone replacement therapy poses a specific thrombotic risk to women. Hormone replacement therapy contains estrogen and is combined with a progestogen in women who still have their uterus. Hormone replacement therapy during menopause is associated with a two- to four-fold increased risk of deep vein thrombosis (6). There is evidence that the thrombotic risk depends on the route of estrogen administration. In a population based study using the data set of about one million women, a higher risk for venous thrombosis was seen in women using oral compared to transdermal hormone

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Venous Thrombosis S10 Novel Antithrombotic Drugs Sam Schulman McMaster University, Clinical Thromboembolism Program Hamilton, Ontario, Canada During the past decade many studies on highly specific, orally available anticoagulants in the treatment of venous thromboembolism (VTE) and for stroke prophylaxis in atrial fibrillation (SPAF) have been published. This story started with the first oral thrombin inhibitor, ximelagatran, but the drug was withdrawn early from the market due to liver toxicity. In 2009 the phase III studies on the next oral thrombin inhibitor were published, showing in comparison with warfarin similar or improved efficacy for SPAF and similar efficacy in treatment of VTE. There was also a reduction of some bleeding outcomes. Subsequently, the pattern has been repeated with oral direct factor Xa inhibitors. The phase III program of the first three of those agents has been fully presented and the drugs has been approved for SPAF in many jurisdictions and one drug, rivaroxaban, also for treatment and for extended secondary prophylaxis of VTE. These new agents appear to provide the same efficacy as the combination of low-molecularweight heparin overlapping with a vitamin K antagonist for the typical patients with deep vein thrombosis and pulmonary embolism. It is therefore anticipated that the new agents will slowly take over the market for this indication. The safety is an important component in this development. All new anticoagulants showed a reduced risk for intracranial hemorrhage compared to vitamin K antagonists in the SPAF studies, and this is probably the same in the smaller venous thromboembolism trials. Intracranial hemorrhage is the most feared complication of anticoagulation and therefore the new agents should lead to a lower resistance against treating patients appropriately for longer periods. Some of the drugs were started in the trials with only one or two initial doses of parenteral therapy, whereas others had a full week of overlap. It is not unlikely that physicians will prefer the initial parenteral therapy for patients with extensive deep vein thrombosis with significant pain and swelling of the leg. Likewise, patients with large pulmonary emboli may also be treated with parenteral therapy in the hospital until they are completely stable. In the extended treatment, typically beyond 6 months trials have assessed low-dose warfarin, new anticoagulants and aspirin. There seems to be a trade-off between efficacy and safety that can be used to tailor the treatment according to the preferences and concerns of each patient. It can thus be anticipated that a patient fearing mostly a recurrence of venous thromboembolism will receive the most effective anticoagulant. Conversely, a patient with primarily fear of bleeding will be treated long-term with an anticoagulant drug with slightly lower efficacy but no increase of bleeding versus placebo or with low-dose acetylsalicylic acid if there is an increased risk for arterial thromboembolism. If the anticoagulant treatment will be extended indefinitely, the risk for recurrent venous thromboembolism will be reduced and thereby reasonably also the postthrombotic syndrome, with becomes very burdensome and costly for some patients. Progress in this field may reduce days off from work and treatment expenditures for management of venous ulcers. Special patient groups that demonstrate extreme hypercoagulability were hardly included in these studies. Thus, patients with active cancer constituted only 4-6% of the study

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populations. There has not been any signal that the new anticoagulants are less effective than vitamin K antagonists in this subset. The standard treatment for patients with active cancer and thrombosis is, however, low-molecular-weight heparin for 3-6 months and the new anticoagulants should be evaluated against this comparator to convince prescribing physicians. Furthermore, patients with antiphospholipid syndrome can also be very hypercoagulable and need to be studied with the new agents. A concern with the new and very convenient new anticoagulants is that family practitioners may bypass the diagnostic imaging in case of high degree of suspicion and directly prescribe an oral thrombin- or factor Xa inhibitor. Another issue is that non-hematologist physicians may not take their time to explain to the patients the rationale for anticoagulant treatment, the potential consequences of poor compliance and actions to take in case of side effects. If the result is that the patients at an early stage drop the anticoagulant treatment, the result will be an increase of the recurrent thromboembolic events. Education and educational tools is therefore key for the success of these new agents. 

Recommended reading: 1.

Weitz JI, Eikelboom JW, Samama MM. New antithrombotic drugs: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012;141:e120S-51S.

2.

Connolly SJ, Ezekowitz MD, Yusuf S, et al. Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med. 2009;361:1139-1151.

3.

Schulman S, Kearon C, Kakkar AK, et al. Dabigatran versus warfarin in the treatment of acute venous thromboembolism. N Engl J Med. 2009;361:2342-2352.

4.

Wolowacz SE, Roskell NS, Plumb JM, et al. Efficacy and safety of dabigatran etexilate for the prevention of venous thromboembolism following total hip or knee arthroplasty: a metaanalysis. Thromb Haemost. 2009;101:77-85.

5.

Patel MR, Mahaffey KW, et al. Rivaroxaban versus warfarin in nonvalvular atrial fibrillation. N Engl J Med. 2011;365:883-91.

6.

Bauersachs R, Berkowitz SD, Brenner B, et al. Oral rivaroxaban for symptomatic venous thromboembolism. N Engl J Med. 2010;363:2499-510.

7.

Buller HR, Prins MH, Lensing AW, et al. Oral rivaroxaban for the treatment of symptomatic pulmonary embolism. N Engl J Med 2012;366:1287-97.

8.

Granger CB, Alexander JH, McMurray JJ, et al. Apixaban versus warfarin in patients with atrial fibrillation. N Engl J Med. 2011;365:981-92.

9.

Connolly SJ, Eikelboom J, Joyner C, et al. Apixaban in patients with atrial fibrillation. N Engl J Med. 2011;364:806-17.

10. Agnelli G, Buller HR, Cohen A, et al. Oral apixaban for the treatment of acute venous thromboembolism. N Engl J Med. 2013;369:799-808. 11. Giugliano RP, Ruff CT, Braunwald E, et al. Edoxaban versus warfarin in patients with atrial fibrillation. N Engl J Med. 2013;369:2093-104. 12. The Hokusai-VTE Investigators. Edoxaban versus warfarin for the treatment of symptomatic venous thromboembolism. N Engl J Med. 2013;369:1406-15.

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Venous Thrombosis S11 How to Approach a Patient with Confirmed Venous Thrombosis Bernd Pötzsch Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn Bonn, Germany Once the diagnosis of venous thrombosis has been established and initial anticoagulant treatment has been started using low molecular weight heparin or rivaroxaban/epixaban the physician is faced with the question how long the anticoagulant therapy should be continued and which type of oral anticoagulant should be used. In patients developing a deep venous thrombosis (DVT) during a typical risk situation such as surgical intervention the majority of guidelines such as the 2012 American College of Physicians Evidence-based Clinical Practice Guidelines recommend anticoagulant treatment for 3 months. There is further consensus that patients developing DVT outside a typical risk situation will benefit from extended anticoagulant treatment. However, it is still a matter of debate if those patients should be tested for endogenous thrombophilic risk factors including APC resistance/FV-Leiden mutation, protein C/S, antithrombin, prothrombinG20210A-mutation, lupus anticoagulant/antiphospholipid antibodies and PNH. Although it has been shown that the development of unprovoked DVT by itself indicates a high risk of recurrence and therefore justifies extended anticoagulant treatment, the results of the thrombophilia screen give further information on the overall risk situation and might be helpful in tailoring the duration of the anticoagulant treatment. For example, patients tested positive for antiphospholipid antibodies should receive anticoagulant treatment until stable remission of the antiphospholipid antibodies occurs but do not require life-long anticoagulant treatment. Furthermore, relatives carrying the same mutation as the index patient might benefit from thromboprophylaxis when undergoing typical risk situations. Another important question is that of testing for occult cancer. Although unprovoked thrombosis is a classical symptom of a paraneoplastic syndrome there is no evidence that patients benefit from an extensive cancer search. Therefore it is recommended to restrict the cancer search to patients with other cancer symptoms such as unexplained weight loss, fatigue, fever, etc.. As randomized clinical trials focused on the anticoagulant treatment of thrombosis at uncommon sites are lacking recommendations of the anticoagulant treatment of thrombosis of the cerebral veins and sinus, gastrointestinal tract, portal veins and renal veins are based on personal experience and retrospective studies. Pros and cons of long-term anticoagulant treatment in these patients will be discussed. Two cohorts of patients represent major treatment challenges. These are thrombosis patients with underlying diseases that predispose them to a high risk of bleeding and patients who develop thrombosis while under oral anticoagulant treatment. Typical examples of patients who are at high risk of bleeding are patients with ulcerative colitis. In those patients the use of oral anticoagulants is associated with a high bleeding risk and parenteral anticoagulants such as low molecular weight heparins or fondaparinux are preferred. Patients developing thrombosis while under treatment with anticoagulants should undergo an extended screening for an underlying malignant disease. A d-dimer based algorithm for increasing the anticoagulant intensity in those patients will be discussed.

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Finally the management of thrombosis in childhood will be discussed, although compared to adults, venous thrombosis in children are relatively uncommon. In most children the venous thrombosis result from secondary complications of primary underlying diseases such as infection, cancer, congenital heart disease, inflammatory conditions or are related to therapeutic interventions such as central venous catheters.  Recommended reading: 1.

Ageno W, Gallus AS, Wittkowsky A, Crowther M, Hylek EM, Palareti G. Oral anticoagulant therapy – antithrombotic therapy and prevention of thrombosis, 9th ed: American College of Chest Physicians evidence-based clinical practice guidelines. Chest 2012; 141: e44S – e88S

2.

Kearon C, Akl EA, Comerota AJ, Prandoni P, Bounameaux H, Goldhaber SZ, Nelson ME, Wells PS, Gould MK, Dentali F, Crowther M, Kahn SR. Antithrombotic therapy for VTE disease – Antithrombotic therapy and prevention of thrombosis, 9th ed: American College of Chest Physicians evidence-based clinical practice guidelines. Chest 2012; 141: e419S-e494S

3.

Kleinjan A, Aggarwal A, van de Geer A, Faselis C, Büller HR, Di Nisio M, Rickles FR, Kamphuisen PW. A worldwide survey to assess the current approach to the treatment of patients with cancer and venous thromboembolism. Thromb Haemost 2013; 110: 959-965

4.

Lee AY, Peterson EA. Treatment of cancer-associated thrombosis. Blood 2013; 122: 23102317

5.

Goodin S. Selecting an anticoagulant for recurrent venous thromboembolism in cancer. Am J Health Syst Pharm 2005; 62: S10-S13

6.

Liew A, Eikelboom JW, O´Donnell M, Hart RG. Assessment of anticoagulation intensity and management of bleeding with old and new oral anticoagulants. Can J Cardiol 2013; 29: S34-S44

7.

Cardiovascular disease educational and research trust, European venous forum, north American thrombosis forum, international union of angiology, union international du phlebologie. Prevention and treatment of venous thromboembolism: international consensus statement (guidelines according to scientific evidence). Clin Appl Thromb Hemost 2013; 19: 116-118

8.

Liew A, Douketis J. Initial and long-term treatment of deep venous thrombosis: recent clinical trials and their impact on patient management. Expert Opin Pharmacother 2013; 14: 385-396

9.

Schulman S. Optimal duration of anticoagulant therapy. Semin Thromb Hemost 2013; 39: 141-146

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Venous Thrombosis S12 Perioperative Management in Patients with Risk for Thrombosis Sabine Eichinger Medical University of Vienna, Department of Internal Med Vienna, Austria Patients at risk of thrombosis who need to undergo surgery or other invasive procedures are not only at an increased risk of thrombosis but also of bleeding. The thrombotic risk is composed by the patient’s intrinsic risk and the risk that is specific for the respective procedure. In analogy, the bleeding risk is influenced by patient characteristics and the type of surgery. Thus, for optimal perioperative management the thrombotic risk needs to be weighed against the bleeding risk.

Postoperatively, low molecular weight heparin at thromboprophylactic dose should be given except in patients undergoing total hip- or knee replacement in which case direct oral anticoagulants are licensed also for postoperative thromboprophylaxis. Oral anticoagulants can be started once hemostasis has been achieved and the surgeon considers it as safe. Notably, the maximum anticoagulant effect of direct oral anticoagulants is reached already two hours after administration. 

Invasive procedures with low bleeding risk (including tooth extractions and other dental procedures, minor dermatologic surgical interventions, endoscopy with low bleeding risk, cataract surgery) should be performed without discontinuing of anticoagulant treatment (1). Of note, in case of treatment with a vitamin K antagonist the international normalized ratio (INR) should be around 2.5 (not higher than 2.8) on the day of the procedure. Patients who are treated with a direct oral anticoagulant should take the medication after the procedure rather than before.

Recommended reading:

Patients who undergo surgical interventions with a higher bleeding risk (including, organ biopsies, thoracal, urogenital or abdominal surgery) need to stop their anticoagulant treatment. Because of their long half-life vitamin K antagonists must be discontinued already several days before surgery which then can be safely performed once the INR is below 1.5 (2). Administration of vitamin K will enhance the normalization of the INR. Requirement of perioperative bridging with low molecular weight heparin depends on the patient’s thrombotic risk. In patients with low thrombotic risk (low risk atrial fibrillation, mechanic heart valve in aortic position without risk factors, VTE more than 12 months ago) preoperative heparin bridging should not be performed. All other patients should receive low molecular weight heparin at therapeutic (or half therapeutic doses in case of increased bleeding risk) already before surgery. Heparin at therapeutic doses needs to be stopped 24 hours before intervention and should not be given before 48 hours after surgery because of an otherwise increased bleeding risk. In a metaanalysis the risk of major bleeding in patients receiving heparin bridging was almost 4 times higher (Odds Ratio 3.60; 95% CI 1.52– 8.50) compared to the non-bridged cohort (3). As there was no difference in thromboembolic complications between the two groups, heparin bridging should be performed with caution and only in moderate and high risk patients.

1. Douketis JD, Spyropoulos AC, Spencer FA, Mayr M, Jaffer AK, Eckman MH, Dunn AS, Kunz R; American College of Chest Physicians. Perioperative management of antithrombotic therapy: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines.Chest. 2012;141(2 Suppl):e326S50S 2.

Spyropoulos AC, Douketis JD. How I treat anticoagulated patients undergoing an elective procedure or surgery. Blood. 2012;120:2954-62.

3.

Siegal D, Yudin J, Kaatz S, Douketis JD, Lim W, Spyropoulos AC. Periprocedural heparin bridging in patients receiving vitamin K antagonists: systematic review and meta-analysis of bleeding and thromboembolic rates. Circulation. 2012 ;126:1630-9.

4.

Wysokinski WE, McBane RD 2nd. Periprocedural bridging management of anticoagulation. Circulation. 2012;126:486-90.

5.

Weltermann A, Brodmann M, Domanovits H, Eber B, Gottsauner-Wolf M, Halbmayer WM, Hiesmayr JM, Kyrle PA, Längle F, Roithinger FX, Watzke H, Windhager R, Wolf C, Zweiker R. Dabigatran in patients with atrial fibrillation: perioperative and periinterventional management. Wien Klin Wochenschr. 2012;124:340-7

Because of their short half-life, perioperative management of direct oral anticoagulant is less complex compared to vitamin K antagonists (4). In case of direct factor Xa inhibitors (rivaroxaban, apixaban), treatment should be interrupted 24 hours before surgery in patients without impaired renal function. The direct thrombin inhibitor dabigatran is primarily cleared by the kidneys. The time of discontinuation of the drug before an invasive procedures depends on the renal function of the patient and the bleeding risk and ranges between 24 hours and up to 5 days (in case of a creatinine clearance of less than 50 ml/ min and a surgery with high bleeding risk) (5). Preoperative bridging with low molecular heparin is required for none of the direct oral anticoagulants.

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Platelets, Platelet Disorders S13 Platelet Function

S14 Heparin-Induced Thrombocytopenia (HIT) Christian Gachet

Ted Warkentin

Inserm, Université de Strasbourg Alsace, France

Hamilton Regional Laboratory Medical Program, Hematology Department Hamilton, Ontario, Canada

The essential role of blood platelets is to maintain vascular integrity and to ensure primary haemostasis, which means cessation of bleeding, upon vascular injury. The main platelet functions required to fulfil this role are 1) adhesion at sites of vascular damage through interaction of major membrane glycoproteins (the GPIb-V-IX complex), the Willebrand Factor circulating adhesive protein, and subendothelial matrix proteins (collagens, laminins, and fibronectin, among others); 2) activation triggered by multiple receptors and signalling pathways including integrins (α2β1, α6β1, αIIbβ3, αvβ3), immunoglobulin-like receptors (GPVI, CLEC-2) and G protein-coupled receptors for ADP (P2Y1, P2Y12), thrombin (PAR1, PAR4), thromboxane A2, serotonin, and adrenaline; 3) secretion of various alpha and dense granule contents such as adhesive proteins, coagulation and growth factors, nucleotides and serotonin among others; 4) aggregation via ligation of soluble fibrinogen to the αIIbβ3 integrin; 5) procoagulant activity by exposure of phosphatidylserine where coagulation factors bind and thrombin is generated. Activated platelets also expose proteins which allow interaction with leukocytes and endothelial cells.

HIT is a prothrombotic (RR ~12.0) drug reaction caused by platelet-activating IgG that recognize multimolecular platelet factor 4 (PF4)/heparin complexes. Platelet activation is triggered when the PF4-heparin-IgG complexes cross-link FcγIIa receptors. Thrombotic effects arise from: (a) platelet activation, (b) procoagulant platelet-derived microparticles, (c) monocyte tissue factor expression, (d) endothelial activation (not proven), and (e) neutralization of heparin by PF4 released from activated platelets.

Most of these steps occur almost simultaneously although they can be distinguished and described in a sequential way. The same mechanisms are triggered (involved, put into effect) when a thrombus forms in arteries while on the other end each of these functions can be altered to give rise to haemorrhagic diathesis. Beyond haemostasis and its pathological counterpart thrombosis, platelets are also involved in many other physiological and pathophysiological processes, including tissue repair, angiogenesis, innate and acquired immunity, embryonic development, vascular inflammation, atherosclerosis and metastatic dissemination. The mechanisms involved in these roles are less well known and some of them may be specific such as the role of CLEC2 in the organisation of the lymphatic vasculature during embryogenesis, TLR receptors during sepsis or particular integrins in cancer dissemination. 

The typical clinical picture is an otherwise unexplained drop in the platelet count that begins 5-10 days into an immunizing heparin exposure. About 50-75% develop clinically evident thrombosis, most often deep-vein thrombosis (DVT) ± pulmonary embolism (PE). Less common venous thromboses include: adrenal vein thrombosis (= adrenal hemorrhage, either uni- or bilateral [risk of fatal adrenal failure]), mesenteric vein thrombosis, and cerebral venous dural sinus thrombosis. Upper-limb DVT occurs in association with central venous catheters. Arterial thrombosis most often causes acute limb ischemia (thrombosis of large limb arteries) > stroke > myocardial infarction. Rarely, skin necrosis occurs at sites of subcutaneous (sc) heparin injection. Patients can develop anaphylactoid reactions (fever/chills, hypertension, dyspnea, cardiac arrest, transient global amnesia) with 30min of an IV heparin bolus or within 2h of sc LMWH. About 10-20% of patients develop overt DIC (elevated INR, greatly increased D-dimer, and [relative] hypofibrinogenemia: fibrinogen 4.5 g/L). Although severe HIT-associated DIC can cause acral limb necrosis in a limb with DVT, most HIT-associated venous limb ischemia (gangrene with pulses) occurs due to warfarin. Vitamin K antagonism is the most common cause of limb loss in HIT, due to profoundly disturbed procoagulant-anticoagulant balance: greatly increased thrombin generation (HIT) plus severe protein C depletion (warfarin); patients exhibit a supratherapeutic INR (>3.5; surrogate marker for severe protein C depletion via parallel severe depletion of factor VII). HIT’s immune nature, featuring unusual antibody (Ab) transience, leads to its striking temporal features. HIT begins 5-10 days after into an immunizing course of heparin (first dose = day 0), irrespective of whether this is the first exposure, or whether many previous exposures have occurred. However, if a patient underwent a recent immunizing heparin exposure (within the past 100 days), then restarting heparin can cause abrupt platelet count fall (“rapid-onset HIT”), a profile seen in ~30% of cases. “Delayed-onset” (“autoimmune-like”) HIT features onset (or progression) of thrombocytopenia after heparin has been stopped; patients often have DIC and can fail therapy due to “PTT confounding.” HIT frequency is variable, depending on (a) type of heparin (UFH > LMWH), (b) patient type (surgical > medical > obstetric/pediatric), (c) heparin duration (10 or more days > 5-10 days > less than 5 days), (d) sex (females > males). The role of dosing is complex: although prophylactic-dose (vs therapeutic-dose) heparin may be more immunogenic (due to PF4-heparin stoichiometry), the relationship is confounded by surgical (vs medical) S14 Continued next page

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Platelets, Platelet Disorders S15 Immune Thrombocytopenias S14 Heparin-Induced Thrombocytopenia (HIT) Continued patients being more likely to receive prophylactic-dose heparin. Nevertheless, a postorthopedic surgery female patient receiving 10 days of UFH has HIT risk of ~5%, whereas a patient with a brief, highly-immunizing UFH exposure (cardiac surgery) but without ongoing postoperative heparin exposure has risk 0.02-0.05%, although the course likely will be severe (“delayed-onset HIT”). “Spontaneous” HIT features a clinical and serological picture identical to HIT, except that no proximate heparin exposure is identified. Affected patients are either post-knee replacement (perhaps cartilage glycosaminoglycans substitute for heparin?) or have preceding infection (bacterial cell walls bind PF4 and can trigger anti-PF4/heparin Abs).

Recommended reading: 1.

Krauel K, ... Greinacher A. Platelet factor 4 binds to bacteria – inducing antibodies cross-reacting with the major antigen in heparin-induced thrombocytopenia. Blood 117:1370–8, 2011.

2.

Warkentin TE. Fondaparinux: does it cause HIT? can it treat HIT? Exp Rev Hematol 3:567, 2010.

3.

Warkentin TE. HIT paradigms and paradoxes. J Thromb Haemost 9 (Suppl 1):105-117, 2011.

4.

Warkentin TE, Kelton JG. Temporal aspects of HIT. N Engl J Med 344:1286-92, 2001.

5.

Warkentin TE, Sheppard JI. Serological investigation of patients with a previous history of heparin-induced thrombocytopenia who are re-exposed to heparin. Blood 2014 in press.

6.

Warkentin TE, et al. Fondaparinux treatment of acute HIT confirmed by the serotonin-release assay: a 30-month, 16-patient case series. J Thromb Haemost 9:2389-96, 2011.

7.

Warkentin TE, Greinacher A, et al. Laboratory testing for HIT: a conceptual framework and implications for diagnosis. J Thromb Haemost 9:2498-2500, 2011

The modern concept of HIT is that patients MUST have detectable anti-PF4/heparin antibodies with platelet-activating properties. Although non-PF4-dependent antigens (e.g., IL-8) are proposed, these are not established. PF4-dependent EIAs have 99-100% sensitivity, though specificity is poor (20-50%); strength of optical density (OD) predicts for platelet-activating Abs (OD > 2.00 units ~90% frequency of a positive washed platelet serotonin-release assay [SRA]). Washed platelet activation assays (SRA, HIPA) performed by experienced labs is the gold standard. Many other PF4-dependent assays are becoming available, e.g., particle gel immunoassay, instrumentation-based assays. Treatment principles of strongly-suspected HIT: 2 Do’s (stop heparin, begin alternative non-heparin anticoagulation), 2 Don’ts (don’t give warfarin [give vit K if VKA already given], avoid platelet transfusions, 2 Diagnostics (test for HIT Abs, image for lower-limb DVT). Two general approaches: (a) direct thrombin inhibitors (DTIs; argatroban, hirudin [NB: lepirudin discontinued], bivalirudin, [?dabigatran]), (b) Xa inhibitors (danaparoid, fondaparinux, [?rivaroxaban]). The Author favors Xa inhibitors (danap, fonda), as they (a) have prophylactic/therapeutic dosing regimens, (b) are effective in Ab+ HIT (whereas efficacy data for argatroban in Ab+ HIT is lacking), and (c) avoid PTT confounding (failure of PTT-adjusted anticoagulation due to misleading PTTs in setting of HIT-DIC). Heparin re-exposure despite previous HIT is reasonable, e.g., need for cardiac surgery: (a) show that platelet-activating Abs are no longer present, (b) use UFH only intra-operatively, (c) use a non-heparin agent post-operatively, if needed (e.g., fonda). Recurrent HIT beginning 5-10d post-op is possible if the patient makes Abs that activate platelets without need for heparin (delayed-onset HIT), a scenario that has been reported. 

Andreas Greinacher Institut für Immunologie und Transfusionsmedizin Universitätsmedizin Greifswald, Germany Immune mediated thrombocytopenias are a heterogeneous group of platelet disorders caused by antibody- or T-lymphocyte-mediated platelet destruction. The underlying cause can be autoimmune disease (autoimmune thrombocytopenia [ITP]), alloantibody mediated (neonatal alloimmune thrombocytopenia [NAIT] and transfusion induced alloimmune thrombocytopenia), a mixed form with both characteristics (post transfusion purpura [PTP]), or drug dependent. Drug induced thrombocytopenias can be distinguished into several distinct groups with different pathogenesis: drug-dependent immune thrombocytopenia (dITP; e.g. by quinine), GPIIbIIa inhibitor induced thrombocytopenias (fibans and abciximab), hapten induced (e.g. penicillin) and heparin-induced thrombocytopenia (HIT). Rarely drugs (e.g. gold) can induce platelet autoantibodies and thereby trigger ITP. Diagnostic approaches Tests for platelet antibodies measure free antibodies in patient serum/plasma or plateletbound antibodies. With the exception of HIT (and post transfusion purpura), laboratory diagnosis of immune mediated thrombocytopenias is limited due to the relatively low sensitivity of the available tests. ITP: Platelet autoantibody testing has not been endorsed in recent evidence-based guidelines of the management of ITP patients due to low sensitivity of the assays (~60% for chronic ITP; for T-cell mediated platelet destruction no practicable assay is currently available). However, an evolving theme is that the ‘ITP syndrome’ is a disorder which may comprise groups of patients with distinct clinical and serological profiles. To better understand the diagnostic, prognostic and pathogenic role of platelet autoantibodies in ITP, further systematic evaluation is required. Direct tests for platelet autoantibodies, which measure antibodies bound to patient’s platelets, have higher sensitivity than indirect tests, which measure free antibodies in plasma or serum. Assays assessing glycoprotein-specific antibodies (e.g. against against GPIb/IX/V and GPIIb/IIIa), like MAIPA or monoclonal antigen capture EIA (MACE) are much more specific than assays measuring platelet-associated IgG e.g. by flow cytometry. Assays using platelet eluates are commercially available. They may improve the sensitivity of the laboratory diagnosis of ITP, but require further validation. Alloimmune thrombocytopenias: the assays of choice measure platelet alloantibodies by indirect glycoprotein specific assays in the patient’s plasma/serum, or, in case of NAIT, antibodies in the maternal blood. Assays measuring antibody binding to whole platelets have a lower specificity (false positive signals caused by HLA antibodies) and sensitivity (antibodies against antigens with low copy numbers). Some alloantibodies are of low affinity, although they can cause significant platelet destruction in vivo. These low affinity antibodies are washed away during the washing steps in the current EIAs but can be detected by techniques not requiring washing such as surface plasmon resonance. dITP and GPIIb/IIIa inhibitor induced antibodies: drug dependent antibodies are measured in the patient’s plasma/serum by indirect assays using whole platelets in the presence or absence of the drug. The read out is antibody binding measured by EIA or flow cytometry. Continued next page

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Platelets, Platelet Disorders S15 Immune Thrombocytopenias Continued These assays require appropriate controls, as the drug itself can cause false positive and false negative results by interfering with the assay reagents. It is important to add the drug to all assay steps, including to the washing solution, as the labile complex between drug, platelet glycoprotein and antibody will dissociate if the concentration of the drug becomes too low. In several cases not the pharmaceutical form of the drug but a metabolite causes the immune reaction. This is a major limitation for laboratories, as the metabolites are usually not available in the laboratory. Pathogenesis ITP is not a single disease. Rather it describes several different causes which result in autoimmune mediated decrease of the platelet count. Increased platelet destruction resulting in shortened platelet survival is a well-accepted cause of the low platelet count in ITP. Beside classic antibody mediated platelet phagocytosis in the reticuloendothelial system, complement activation may lead to intravascular lysis of platelets (or CD35mediated phagocytosis), and impaired platelet production may also contribute to the low platelet count. Megakaryocytes cultured in plasma of ITP patients show abnormal growth and increased apoptosis, and primed T-cells may directly attack the megakaryocytes. Another relatively newly evolving observation is the increased prevalence of arterial and especially venous thrombosis in ITP patients compared to the normal population. The reasons are not well understood. Intravascular destruction of platelets and resulting platelet microparticles may be one reason. In secondary ITP (see below) also indirect effects caused by the underlying disease may increase the risk for thrombosis. ITP can occur in an acute and transient form or in a chronic form in which thrombocytopenia persists for >12 months. Acute ITP is often triggered by viral infections which, especially in children, typically precede the onset of bleeding symptoms by 1-2 weeks. Although platelet counts often decrease to very low values (